1. Field of the Invention
This invention relates to a split feed fed batch fermentation system for culturing submerged host cells that produce a compound of interest. More specifically, the invention concerns a method whereby organic carbon and nitrogen nutrient sources for the cultured cells are maintained as separate media solutions and are fed independently into a fermentor containing cultured host cells.
2. Description of the Related Art
Production of Pharmaceutical Compounds of Interest
Compounds of interest to the pharmaceutical industry such as certain organic compounds, proteins and carbohydrates, can be produced in large quantities by culturing cells (typically referred to as "host cells") in a liquid nutrient solution. These host cells have either been engineered to produce such compounds, or they produce the compounds naturally. Typically, the host cells are submerged in a tank (often referred to as a "fermentor") containing a liquid nutrient solution (medium) for a set period of time. During culturing or fermentation, the cells grow, multiply, and synthesize the compound of interest. The compound can then be collected either from the culture medium, or by harvesting the cells and extracting the compound directly from the cells.
Many different species and strains of host cells, either aerobes or anaerobes, can be used for preparation of compounds of interest. Selection of the host cell species is usually dependent on the nature of the compound to be produced. For example, heavily glycosylated recombinant proteins are typically produced in eukaryotic host cells. A commonly used eukaryotic cell line is a Chinese hamster ovary cell line (CHO cell line). Yeast or fungal cells such as Streptomyces are useful hosts for making certain recombinant polypeptides that are lightly glycosylated, as yields of the polypeptide from yeast cells can be greater than yields from mammalian cells. Bacterial cells such as E. coli or Bacillus are often preferred for manufacturing non-glycosylated polypeptides. Bacterial cells are generally considered to be the easiest cell type to culture, as their nutritional requirements are relatively simple, their growth rate is higher, and they can be grown to relatively high densities in a fermentor as compared to eukaryotic cells.
Commercial-scale fermentation has been used successfully to prepare several human recombinant polypeptide pharmaceutical compounds such as, for example, insulin, erythropoietin, granulocyte-colony stimulating factor (G-CSF), tissue plasminogen activator (t-PA), and human growth hormone (hGH), as well as many other polypeptides.
Fermentation Systems
A variety of fermentors and fermentation systems exist for submerged culturing of cells, and selection of a suitable fermentation system is dependent on a number of factors such as, for example, the amount of the compound to be produced, the host cell species to be employed, whether the compound is secreted by the host cells, the duration of fermentation, and the resources and capital available.
Two commonly used fermentation systems are the continuous culture system and the fed batch culture system. The continuous culture system is typically used to extend the growth phase of the cultured cells over long periods of time by providing fresh medium to the cells while simultaneously removing spent medium and cells from the fermentor. Such a culturing system serves to maintain optimal culturing conditions for certain cell types and products, i.e., constant volume of medium in the fermentor, constant cell concentration in the fermentor, and constant product concentration in the fermentor. Steady-state maintenance of cell density is accomplished by providing sufficient levels of all required nutrients to the cells.
Fed batch fermentation systems are generally defined as batch culture systems wherein fresh nutrients and/or other additives (such as precursors to products) are added but no medium is withdrawn. In one type of fed batch fermentation system, the fermentation period is divided into two phases, a growth phase, and a production (or synthesis) phase. The growth phase, which commences upon introduction (inoculation) of the host cells into the fermentor, is the time period during which the host cells grow and divide, thereby increasing the host cell density. The length of this phase is primarily a function of the type of host cells being cultured and the rate at which they multiply. After the host cells have achieved the desired density in the fermentor, the fermentation conditions can be altered such that the host cells are induced to produce the compound of interest (the production or synthesis phase).
This type of fed batch fermentation system is often appropriate for cells that can produce a compound in a manner that is decoupled from their growth, as is the case for many polypeptides produced using recombinant DNA technology. For example, production of many heterologous polypeptides in bacterial host cells is typically independent from the growth of the cells. In these cells, the maximum amount of product is often produced by growing the cells to a certain density, and then inducing the cells (by addition or removal of certain compounds or by changing the temperature of the culture medium, for example) to synthesize large quantities of polypeptide. Once the amount of the compound produced by the cells levels off, the cells, medium, or both can be harvested to obtain the compound of interest.
The fermentors used in fed batch culture systems often have probes attached. The probes can monitor various parameters during the fermentation process. The probes may be attached to a computer. Such parameters as dissolved oxygen, optical density, respiratory quotients, pH, temperature, and the concentration of such toxic compounds produced by the host cells such as acetate can be monitored either directly by the probes, or by removing samples of the culture medium and analyzing them off-line using standard assays. By monitoring these parameters, the optimal time to change the medium from the growth medium to the production medium can readily be identified.
The selection of the growth phase and production phase media to be used in a fed batch culturing system is primarily a function of the nutritional requirements of the host cell line used, the rate of growth of the cells, and the chemical composition of the compound to be produced. Often, the organic nitrogen content of the medium will be different for growth phase and production phase media, especially where the compound to be produced is a protein. For example, Tsai et al. (J. Indust. Microbiol., 2:181-187 [1987]) cultured E. coli cells engineered to produce the polypeptide IGF-1 in a fed batch culture system. They found that the yield of IGF-1 increased if the amount of organic nitrogen was increased in the production phase medium. Jung et al. (Miami Bio/Technol. Winter Symp., 8:60 [1988]) found that an increased level of yeast extract (a standard source of organic nitrogen) in the medium added to the E. coli host cells during the production phase resulted in an increased production level of the polypeptide IL-1 beta by the cells.
Certain problems with the preparation of fermentation media have been routinely encountered. For example, it is believed that some sugars or carbohydrates can form covalent complexes with organic phosphate groups during heat sterilization. Thus, these two components of many media are not generally heat sterilized together. Instead, it is generally preferred to sterilize them in separate solutions, and then recombine them at a lower temperature to form the completed medium.
Another problem commonly encountered in preparing fermentation media is the formation of precipitates either before, during, or after sterilization, which can be due to over saturation of the solutions.
Producing biological compounds by fermentation, especially fed batch fermentation, is an expensive procedure, as the demand for purified water and clean steam (for sterilization) can be high, the process is generally labor intensive, requiring skilled workers at all levels of production, and the cost of high grade nutrients and other ingredients necessary for culture media can be high.
There is thus a need in the art to provide a simple fed batch fermentation system that minimizes the cost for production of a compound of interest such as a polypeptide by 1) reducing the number of tanks and sterile media transfers necessary for the fermentation process; 2) providing required nutrients to cells in a more timely and cost effective manner so as to minimize cost and maximize yield of the compound of interest; and 3) reducing problems that may occur in media preparation such as precipitation of certain ingredients or the formation of chemical intermediates during sterilization.
Accordingly, it is an object of the present invention to provide a fermentation system that reduces the cost of production of biological compounds, decreases the number of tanks to be sterilized, and provides nutrients to the cultured cells in a more cost efficient manner as compared with known methods.